This invention relates to chemical and biochemical assays, and more particularly to such assays wherein the volume, relative to a given sample volume, of at least one of the constituent phases of a fluid suspension is to be quantitated without physically separating the phases from one another.
A problem frequently encountered in the assay of fluid suspensions is to determine the volume, relative to the total volume, of one or more of the constituent phases. Thus, for instance, in the assay of milk, it is frequently desired to determine the percentage butterfat content. As another example, in the assay of whole blood, the percentage cellular volume [generally expressed as a packed cell volume (PCV) or hematocrit] is frequently desired. Such relative volumes are commonly determined after the constituent phases of the suspension are physically separated from one another, as by centrifugation.
Not only is such a separation step a typical preliminary to the quantitation of the constituent phases of a suspension, it is also generally a preliminary to yet other assays. Thus, to consider again the assay of blood, a large number of protocols require a serum sample, the blood first being centrifuged to separate from the serum the approximately 50% volume of cells. As a consequence, any protocol permitting the use of whole blood requires a correction for cell volume to relate the whole blood measures to the serum measures that constitute the accepted body of clinical data. Similar considerations occur with respect to the assay of a constituent phase of other suspensions.
A particular class of assays that may incorporate the present invention are immunoassays. Such assays, in which aliquots of sample and one or more reagents are variously reacted to form antigen-antibody or similar complexes which may then be observed in order to assay the sample for the presence and titer of a predetermined moiety of the complex, are well known. Typical of such assays are those wherein a specific antibody is used to measure the quantity of the antigen for which it is specific (or vice versa). However, the technique has been extended to quantitate haptens (including hormones, alkaloids, steroids, and the like) as well as antigens, and antibody fragments (i.e., Fab) as well as complete antibodies, and it is in this broader sense that the present invention should be understood.
As is well known, sensitive immunoassays employ tracer techniques wherein a tagged constituent of the complex is incorporated into the reagent, the non-complexed tagged reagent being separated from the complexed reagent, and the complex (or non-complexed reagent) then quantitated by observing the tag. Both radioisotopes and fluorescent markers have been used to tag constituents of immunoassay reagents, the tag being respectively observed by a gamma ray counter or a fluorimeter. The present invention is, however, directed only to those assays which rely on fluorescence.
The separation of the non-complexed tagged moiety from the complexed is commonly accomplished by immobilizing a predetermined one of the components of the complex to a solid phase (such as the inside wall of a test tube, glass or polymeric beads, or the like) so as hopefully not to hinder the component's reactivity in forming the complex. As an example, an antibody such as immunoglobulin G (IgG) may be bound to a solid phase, such as glass, by a silyl compound such as 3-aminopropyltrimethoxy silane, through the use of bifunctional reagents such as phenyl diisothiocyanate. Any complex formed incorporating the immobilized component may then be physically separated from the non-reacted complement remaining in solution, as by aspirating or decanting the fluid from a tube or by eluting the fluid through a particulate bed. Alternatively, as disclosed, for instance, in copending U.S. patent application Ser. Nos. 406,324 (filed Aug. 9, 1982) and 410,340 (filed Aug. 23, 1982), total reflection fluorescence (TRF) and fluorescence tunnelling may be used to restrict the excitation and observation of fluorescence to an extremely thin layer adjacent the solid phase, thereby in effect accomplishing the separation of the immobilized tagged component from that remaining in solution by optical means.
The methods so far described are applicable to a number of immunological procedures. In competition immunoassay, for instance, the reagent consists of a known quantity of tagged complement (such as antigen) to the immobilized component of the complex (in this instance, antibody). The reagent is mixed with a fixed quantity of the sample containing the untagged complement to be quantitated. Both tagged and untagged complement attach to the immobilized component of the complex in proportion to their relative concentrations. After incubation for a set time, the fluid sample and reagent are separated. The complex immobilized to the solid phase is then illuminated with radiation of a wavelength chosen to excite fluorescence of the tag, and the fluoresence is measured. The intensity of the fluoresence of the immobilized complex is inversely proportional to the concentration of the untagged complement being assayed.
Alternatively, an assay may be made by immobilizing a quantity of an analog of the moiety to be quantitated (i.e., a substance which is immunologically similarly reactive) and reacting the sample with a known quantity of tagged complement. The tagged complement complexes with both the unknown quantity of the moiety in the sample and the immobilized analog. Again, the intensity of fluorescence of the immobilized complex is inversely proportional to the concentration of the (free) moiety being quantitated.
So-called "sandwich" immunoassays may be performed for multivalent complements to the immobilized component, the attached complement being then further reacted with a tagged analog of the immobilized component. Thus, bivalent antigen may be bound to an immobilized antibody and then reacted with a fluorescent tagged antibody, forming an antibody-antigen--tagged antibody sandwich that may then be separated from the unreacted tagged antibody. The intensity of the fluorescence of the thus formed immobilized complex is directly proportional to the concentration of the species being quantitated.
A number of problems arise in the fluorescent assay of biological fluids. Thus, for instance, erythrocytes are highly absorbing. Accordingly, prior art fluorescent assays of blood typically centrifuge the sample and assay the supernatant serum. Typical centrifugation procedures require on the order of 15 to 30 minutes, a time period that may be inconsistent with some clinical procedures where assay results are required rapidly (so-called "stat" procedures). For such cases, state-of-the-art microsampling techniques and centrifuges do permit serum extraction in less than a minute; however, such centrifuges are not, as yet, available in all clinical laboratories, and in any event, serum extraction, by whatever means, is an additional step, and consequently a potential source for the introduction of human error. Further, means for serum extraction are not available in all settings where a rapid assay may be desireable (e.g., mobile emergency facilities, home care, and the like).
The total reflection fluorescence assay methods, as described in the aforementioned Ser. Nos. 406,324 and 410,340, by sampling an extremely thin layer of the sample, permit assays of optically dense samples. While in principle this would allow fluorescent assay of whole blood, clinical experience has generally been with serum samples, and consequently, to relate whole blood assays to the accepted body of experimental data requires a quantitation of the serum volume of the sample. A similar consideration occurs with other biological fluids, such as saliva, wherein the sample as collected is also typically a disperse system with an unknown ratio of suspended-to-suspending material (e.g., for saliva, an unknown ratio of bubble to fluid volumes).